Mutations in amelogenins cause enamel defects referred to as amelogenesis imperfecta (AI). Further understanding of how the different amelogenin isoforms are processed in the enamel matrix during secretion will help elucidate the AI disease mechanism. The goal of the proposed research is to investigate the functional importance of processing of the C-terminus of leucine rich amelogenin peptide (LRAP) in enamel formation, and how LRAP interacts with full-length amelogenin (M180) to form enamel prisms. I have selected Carolyn Gibson, an expert in molecular biology and generation of amelogenin AI mouse models, as my primary mentor, and Pamela DenBesten, an expert in enamel protein biochemistry and ameloblast cell culture as my co-mentor. For the K99, mentored phase, the first specific aim is determine how the C-terminus of leucine rich amelogenin peptide (LRAP) is involved in enamel prism formation. Using a transgenic mouse model overexpressing LRAP and M180 with a truncated C-terminus, I will analyze the enamel phenotype and receive additional training in mineral analysis and imaging to determine if the C-terminus of LRAP can rescue the non-prismatic enamel phenotype in mice lacking the full-length amelogenin C-terminus (CTRNC). To address this aim using an in vitro approach, I will receive training in ameloblast cell culture and transfection, mass spectrometry, recombinant protein generation, calcium binding, protein assembly and mineral induction. Using prior training and my training from the K99 phase, the second aim, which will be carried out during the R00 phase, is to determine whether processing of the C-terminus of LRAP by Mmp20 is required during secretory enamel formation. Using transgenic mouse model overexpressing LRAP but lacking Mmp20, I will investigate enamel structure and cleavage products. I will also use recombinant proteins and cell culture to determine whether mutation of the Mmp20 cleavage site at the C-terminus of LRAP can affect mineral binding and formation or nanosphere assembly. Finally, the third specific aim which will be carried out during the independent R00 phase is determine how LRAP, M180 and their cleavage products work together to guide enamel mineral formation during secretory enamel formation. I will use the in vivo and in vitro approaches of transgenic mouse models, nanoindentation, recombinant proteins and cell culture to determine whether M180, LRAP, and truncated M180 and LRAP together can improve enamel formation in vivo, or protein assembly and mineral induction in vitro. The data generated from the proposed experiments can lead to proposition of a mechanism of how the amelogenin isoforms and their cleavage products are involved in enamel mineralization. PUBLIC HEALTH RELEVANCE: Mutations in amelogenins cause enamel defects referred to as amelogenesis imperfecta (AI). Improved understanding of the role of the genes involved in enamel formation can improve the prospects for treatment of children with this condition. The fundamental goal of this research is to determine how abundant amelogenin isoforms are processed and involved in enamel mineral formation, which will help elucidate AI disease mechanisms.